Kinetex c18 100a column

The HPLC system consisted of a gradient pump unit, a thermostated auto sampler, a column oven, a diode array spectrophotometric detector (DAD) and a fluorescence detector (FLD), all Ultimate 3000 Series instruments from Dionex (Sunnyvale, CA, USA), column of Kinetex C18 100A column (Phenomenex, USA) with a security guard KJO-4282 from Phenomenex (Torrance, CA, USA). The fluorescence was recorded at the wavelength of Ex320/Em430 nm and measurement interval of 0.5 s. Chromatographic data was processed with Chromeleon 7.1 software by Dionex Thermo Scientific (Waltham, USA).
The micrOTOF-Q II instrument by Bruker Daltonik GmbH (Bremen, Germany) with ESI source was used for mass-spectrometric analyses. For the identification of 4-PA both positive and negative ion mode were used. Sample analysis were done with the following parameters: mass range of 60–1700 m/z, ion source temperature of 200°C, ESI voltage of 4.5 kV, ESI nebulization gas flow of 8.0 L/min, drying gas flow of 1.2 bar, detector voltage of 2.03 kV and acquisition rate of 1 Hz. Mass calibration was performed with sodium formate solutions from m/z 60 to 1700. For data acquisition software Compass HyStar version 3.2 and for processing Compass DataAnalysis version 4.0 SP1 was used (both Bruker, Billerica, USA).

The glucose, fructose, glycerol, acetate, and ethanol concentrations of the hydrolysis and fermentation samples were analyzed using HPLC (HPLC 1260 series, Agilent Technologies, USA) equipped with a Rezex-ROA Organic Acid H+ column (8%, 150 mm × 4.6 mm; Phenomenex Inc., USA). The analytes were eluted with 0.005 N H2SO4 at 0.6 ml/min and 50°C, as previously described [20 (link)].
Sulforaphane was analyzed using HPLC (Waters, Alliance 2796 Separations System), with a 2996 photodiode array detector (Waters) connected to a Kinetex C18 100A column (5 μm, 250 mm × 4.6 mm: Phenomenex Inc.). The filtrate was analyzed by acetonitrile/water (3:7, v/v) isocratic elution at a flow rate of 0.6 ml/min. A UV/Vis spectrophotometer (Optizen NanoQ, Mecasys Co., Korea) at a wavelength of 205 nm was used to detect sulforaphane.

Freeze-dried samples of 1 g were extracted with 70% methanol (10 mL; 30 min, 70 °C). During the extraction process, samples were shaken on a vortex every 3 min, then filtered and centrifuged (10 min, 15,000 rpm). The supernatant was separated, methanol was removed from the mixture by evaporation on a vacuum evaporator. Next, samples were prepared by dissolving in water (1 mL), and the LC-MS analysis was performed using reversed-phase high-performance liquid chromatography (HPLC Shimadzu Prominence-i LC-2030C, Kyoto, Japan) equipped with a PDA detector coupled to a triple quadrupole mass spectrometer (Shimadzu LCMS-8045). Glycosides were separated using the following mobile phase: water with 0.1% TFA (eluent A) and acetonitrile with 0.1% TFA (eluent B). The flow rate was set at 0.25 mL·min⁻¹, and the gradient was as follows: starting at 1% solvent B for 3 min, then reaching 20% up to 20 min, 30% up to 23 min, and 0.1% B at 35 min. Separation was obtained on a Kinetex C18 100A column (100 × 3 mm, 2.6 µm particle size, Phenomenex, Germany). Singrin and neoglucobrassicin were used as external standards for the analysis [49 (link)].

Mycelia of AO-herA and AO-herA-pks5 transformants were inoculated into a solid medium containing polished rice (1 g) and appropriate adenine at 30 °C for 10 days. After extraction with ethyl acetate, the extract was concentrated in vacuo to afford crude extracts. The crude extracts were analyzed by HPLC equipped with an Agilent TC-C18 (250 mm × 4.6 mm) at the following conditions: 0–5 min, 10% B; 5–20 min, a linear gradient 10–100% B; 20–30 min, 100% B (A: H₂O + 0.1% of formic acid, B: CH₃OH + 0.1% of formic acid) at a flow rate of 1 mL/min. Samples were analyzed using a TripleTOF 6600 mass spectrometer (AB/SCIEX, Milford, MA) and an HPLC system (AB/SCIEX). Chromato- graphic separation was achieved using a 150 mm × 4.6 mm, 2.6 μm Kinetex C18 100A column (Phenomenex) at the following conditions: 0-10 min, 5–100% B;10–15 min, 100% B (A: H₂O + 0.1% of formic acid, B: CH₃CN + 0.1% of formic acid) at a flow rate of 0.6 mL/min.

Carotenoid analysis was performed as described by Sadler et al. [22 (link)]. Carotenoids were extracted from 500 mg of dried powder samples, obtained from pericarp tissues, using hexane, acetone, and ethanol (2:1:1). The hexane layer was collected, and the concentrated solution was adjusted to 2 mL (v/v) with methyl tert-butyl ether and filtered for analysis. Carotenoids were quantified using an HPLC Agilent 1200 series system (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a Kinetex C18 100A column (100 × 4.60 mm, 2.6 μm; Phenomenex Inc., Torrance, CA, USA). The HPLC conditions were as follows: column temperature, 40 °C; detection wavelength, 454 nm; flow rate, 0.8 mL/min; and injection volume, 20 μL. Carotenoids were analyzed via gradient elution (70–100%) of mobile phase solvents A (water:methanol = 25:75 (v/v)) and B (ethyl acetate). Compounds were identified by comparing their elution times with those of the verified standards.

The glucose, fructose, glycerol, acetate, and ethanol concentrations of the hydrolysis and fermentation samples were analyzed using HPLC (HPLC 1260 series, Agilent Technologies, USA) equipped with a Rezex-ROA Organic Acid H+ column (8%, 150 mm × 4.6 mm; Phenomenex Inc., USA). The analytes were eluted with 0.005 N H2SO4 at 0.6 ml/min and 50°C, as previously described [20 (link)].
Sulforaphane was analyzed using HPLC (Waters, Alliance 2796 Separations System), with a 2996 photodiode array detector (Waters) connected to a Kinetex C18 100A column (5 μm, 250 mm × 4.6 mm: Phenomenex Inc.). The filtrate was analyzed by acetonitrile/water (3:7, v/v) isocratic elution at a flow rate of 0.6 ml/min. A UV/Vis spectrophotometer (Optizen NanoQ, Mecasys Co., Korea) at a wavelength of 205 nm was used to detect sulforaphane.

Preparative RP-HPLC was run on a Gilson PLC 2250 HPLC system (Villiers le Bel, France) using a preparative column (Waters DeltaPak C18 Radial-Pak Cartridge, 100 Å, 40 mm × 100 mm, 15 μm particle size, flow rate 50.0 mL/min). Buffer A was 0.1% TFA in water, and buffer B was 0.1% TFA in ACN. Fully folded synthetic TxIA ribbon was purified on a UltiMate 3000 UHPLC system (Thermo Fischer Scientific) using an Kinetex C18 100 A column (100 mm × 2.1 mm, 2.6 μm particle size) from Phenomenex (France). Buffer A was 0.1% formic acid in water, and buffer B was 0.1% formic acid in ACN.